Method for identifying modulators of ion channels

ABSTRACT

The present invention provides methods to manipulate differentiation of a neuroblastoma cell line (IMR-32) such that predominant Na v  expression is either Na v 1.3 in IMR-32 cells exposed to retinoic acid or Na v 1.7 in cells grown under non-differentiating conditions. The cells of the present invention are useful for the discovery of new compounds that modulate the function of either Na v 1.3 and/or Na v 1.7.

BACKGROUND OF INVENTION

[0001] Voltage-gated sodium (Na) channels (Na_(v)) are complex integralmembrane proteins that open by depolarization, allowing the influx ofNa⁺ions which, in turn, mediate the fast depolarization phase of anaction potential in many excitable cells, e.g., neurons, neuroendocrinecells, and cardiac and skeletal myocytes. The nine known alphapore-forming Na_(v) subunits that have been functionally expressed areclassified into two major pharmacological groups: Na_(v) that are eitheri) sensitive or ii) insensitive to tetrodotoxin (TTX), a lethal toxinisolated from puffer fish (Fugu. sp). TTX-sensitive (TTX-S) channels areblocked by low nM concentrations of TTX while TTX-resistant (TTX-R)channels are blocked by μM concentrations of TTX. Members of the TTX-Sclass include SCN1a (Na_(v) 1.1), SCN2a (Na_(v) 1.2), SCN3a (Na_(v)1.3), SCN4a (Na_(v) 1.4), SCN8a (Na_(v) 1.6), and SCN9a (Nav1.7).Members of the TTX-R class include SCN5a (Na_(v) 1.5), SCN10a (Na_(v)1.8), SCN12a (Na_(v)1.9) (Clare et al. (2000); Goldin et al. (2000)).SCN6a/SCN7a has not been functionally expressed; however, it ispredicted to be TTX-sensitive since it contains an aromatic amino acid(Y) in the pore region of domain I known to be required for TTX blockade(Akopian et al. (1997)). Na_(v) alpha subunits are very large and sharefeatures with calcium channels and the prototype K_(v) potassiumchannels first described in Drosophila (Fozzard and Hanck (1996)). It isbelieved that channels in this large super-family are formed by theassociation of four similar domains, each with six putativetransmembrane segments (S1-6) and a pore (P) domain. In the case of theclassical Na and Ca channels, these four domains are combined in asingle gene: Domains I-IV (Plummer and Meisler (1999)). Na_(v) alphasubunits form complexes with one or two beta subunits, probably throughcovalent interactions (Isom (2000); Isom (2001)). A variety of toxinshave been shown to bind to other sites on Na_(v) channels, includingsite-2 toxins that bind to site-2 and lead to persistent activation(e.g. veratridine and batrachotoxin). Local anesthetics interact withamino acids in the S6 transmembrane region of domain IV, which, byanalogy to the crystallized K channel KcsA (Doyle et al. (1998)), arethought to line the pore (Clare et al. (2000)).

[0002] From a therapeutic perspective, pharmacological and kineticdifferences of Na_(v) isoforms provide a basis for developingtissue-specific therapeutic agents. For example, some local anestheticagents (e.g. lidocaine) have a greater efficacy in the heart than in thenervous system, while guanidinium and μ-CTX toxins discriminate betweenheart, skeletal and nerve Na channels (Fozzard and Hanck, (1996)). Otherantagonists have been found to block Na channel activity in ause-dependent manner by binding to specific channel conformationspresented in closed, activated or inactivated states. Theseuse-dependent blockers target aberrantly hyperactive channels in certainhuman diseases and thus, can be utilized to assist rational therapeuticdevelopment. However, these are rare examples of Na_(v) subtype-specificantagonists. Fortunately, molecular identification and pharmacologicalcharacterization of channels underlying endogenous Na currents in cellsmay enable the association of specific Na_(v) subtypes to specificdiseases. Aberrant Na_(v) expression has been identified as acontributing factor to human disease and debilitation includingepilepsy, long QT syndrome, and paralysis. Recent investigation hasimplicated aberrant Na_(v) expression as contributing to neuropathicpain (reviewed by Clare et al., 2000). For example, examination ofinjured DRG neurons reveals enhanced expression of certain Na_(v)channels including the TTX-sensitive Na_(v) alpha subunit SCN3a.Following nerve injury, neurons of the Dorsal Root Ganglion (DRG) becomespontaneously active, activate at lower thermal and mechanical stimuliintensities and fire repetitively to supra-threshold stimuli (Gurtu andSmith (1988)). The elevated spontaneous activity in injured DRG can beblocked by local anesthetics (Chabal et al. (1989); Tanelian and MacIver(1991); Devor et al. (1992); Sotgiu et al. (1992); and Matzner and Devor(1994)), a class of compounds known to target Na_(v), as well as TTX(Amir et al. (1999)). In addition, it has been observed that peripheralaxotomy of sensory neurons leads to an increase in a TTX-S sodiumcurrent with a SCN3a-like kinetics, having a significantly fasterrecovery from inactivation (τ˜15 msec) compared to TTX-S sodium currentsin control rat neurons (τ˜60 msec) (Cummins and Waxman (1997)).Noteworthy, in some skeletal muscle Na_(v) channelopathies, includingparamyotonia congenita, an increased rate of Na_(v) recovery frominactivation appears to contribute to the hyperexcitability of skeletalmuscle by reducing the refractory period (Chahine et al. (1996)).

[0003] Delayed hyperexcitability that develops following peripheralnerve injury (thought to underlie some types of “neuropathic pain”)correlates with novel Na_(v) expression including up-regulation of theTTX-sensitive alpha subunit Na_(v) 1.3 (SCN3a) in both unmyelinated andmyelinated sensory neurons (Waxman (1999)). Numerous studies havedemonstrated that peripheral nerve injury increases Na_(v) 1.3expression in rat DRG neurons (Waxman et al. (1994); Black et al.(1999); Dib-Hajj et al. (1999)). For example, intrathecal application ofGDNF reversed the upregulation of Nav1.3 after spinal nerve ligation(method: Kim and Chung, 1992) and attenuated aberrant ectopic activityand neuropathic pain behavior (Boucher et al. (2000)). Relatedly, incultured dissociated small nociceptive DRG neurons, addition of NerveGrowth Factor (NGF) results in down-regulation of SCN3a mRNA (Black etal. (1997)). SCN3a is believed to contribute to neuronalhyperexcitability as a result of its ability to rapidly “reprime”(recovery from inactivation) during the re-polarization phase of theaction potential. For example, in small rat DRG neurons, increasedexpression of Na_(v)1.3 after peripheral axotomy correlated with aswitch from a TTX-S current with slow recovery from inactivation to aTTX-S current with a four-fold more rapid recovery (rapid re-priming),resulting in increased frequency of repetitive firing (Cummins andWaxman (1997)). Physiological properties (e.g. development of andrecovery from inactivation) of the up-regulated Na channel in axotomizedDRG are nearly identical to SCN3a when compared to SCN3a transientlyexpressed in certain cell types (Cummins et al. (2001)). Black andcolleagues showed increased SCN3a immunoreactivity in adult rat smallDRG neurons after axotomy of peripheral sciatic nerve processes but notdorsal rhizotomy (Black et al. (1999)). Furthermore, expression of arapidly-re-priming Na current was restricted to peripherally, notcentrally, axotomized small DRG neurons (Black et al. (1999)).Similarly, Chaplan and colleagues demonstrated by quantitative PCRup-regulation of Na_(v) 1.3 mRNA in lumbar sensory spinal gangliaisolated from diabetic rats and rats treated with the chemotoxic agentvincristine (Chaplan, Calcutt and Higuera, Journal of Pain (2001)2(2):S1:21). Aberrant SCN3a expression following peripheral nerve injuryalso occurs in humans. Coward and colleagues demonstrated SCN3a(Na_(v)1.3) immunoreactivity in a subset of peripheral nerve fibers frompatients that had experienced peripheral or central nerve injury.Consistent with data obtained in rat neuropathic pain models, nodetectable increase in soma labeling was observed after central avulsion(axotomy) in humans (Coward et al. (2001)). Whether SCN3a isup-regulated in human DRG neurons after peripheral axotomy requiresfurther investigation.

[0004] When compared, the kinetic and pharmacologic properties of human(Chen et al. (2000)) and rat (Cummins et al. (2001)) recombinant SCN3achannels are similar. For example, the recovery from inactivation timeconstant is ˜20 msec when membrane potential is held at −90 mV for bothhuman and rat receptors (compare FIGS. 4 and 5 of Chen et al. (2000)with FIG. 4 of Cummins et al. (2001)). The voltage dependence ofactivation and inactivation are similar as well (midpoints of activationwere −23 and −25 mV for human and rat, respectively; half steady stateinactivation potentials were −69 and −65 mV, respectively). Thesimilarity of rat and human SCN3a functional properties supports thehypothesis that increased expression of SCN3a in injured human DRG willlikely contribute to enhanced firing frequencies similar to thoseobserved in injured rat DRG neurons. The beta subunit(s) associated withthe up-regulated SCN3a channel in injured DRG neurons are unknown. Atleast two Na channel beta subunits (β1 and β3) are known to be expressedin DRG neurons (Oh et al. (1995); Coward et al. (2001); Shah et al.(2001)), and it has been reported that in the CCI model of neuropathicpain there is 20% up-regulation of β3 in small diameter DRG neurons(Shah et al. (2001)). Co-expression of β1, β2, β1+β2, or β3 withNa_(v)1.3 revealed that Na_(v) 1.3 voltage dependence of activation wasshifted +7 mV in the presence of only β3. Furthermore, β3 shifted thevoltage dependence of inactivation to the right by +7 mV, and β1+β2 (butneither alone) shifted it by +5 mV (Cummins et al. (2001)). To date, β2has not been detected in cultured rat DRG neurons (Black et al. (1996)).Examined collectively, the aforementioned study data provide strongevidence that over-expression of Nav 1.3 in injured DRG neuronscontributes to the genesis and maintenance of neuropathic pain inanimals, including humans.

[0005] Interestingly, PN1 (also known as SCN9a, hNE (NeuroEndocrinechannel) and Na_(v)1.7) is another TTX-sensitive Na_(v) alpha subunitpreferentially-expressed in rat and human injured DRG neurons,trigeminal ganglion neurons and sympathetic neurons (Toledo Aral et al.(1997)). PN1 has been reported to be up-regulated in small diametersensory neurons up to three months following CFA-induced inflammation ofperipheral receptive fields [England et al., Peripheral Nerve SocietyAbstract (1999)]. In SNS null mice, a 50% up-regulation of PN1 mRNA wassuggested to compensate for the hypoalgesia caused by the absence of SNSin carrageenan-induced inflammation [Akopain et al., Nat. Neurosci,(1999) 2:541]. Examination of injured human DRGs reveals that regulationof PN1 is similar to that of TTX-R channels (Coward et al. (2001)).Furthermore, RT-PCR data suggest a positive correlation betweenup-regulation of Na_(v)1.7 (and Na_(v)1.3) and the metastatic potentialof prostate tumor cell lines (Diss et al. (2001)). Therefore, inhibitorsof Na_(v)1.7 and Na_(v)1.3 may have therapeutic potential in curbingmetastasis in certain cancers including prostate cancer.

[0006] Unfortunately, conventional therapy for treating neuropathic painin humans due to ectopic (spontaneous) Na_(v) activity, includingadministration of analgesics, anticonvulsants and anti-arrhythmics, hasproven sporadically effective with demonstrable side-effects as aconsequence of non-specific, low-potency interactions at Na_(v) targets.This fact, coupled with a growing population of neuropathic painsufferers, reveals the importance and immediate need for Na_(v)subtype-specific antagonists. Historically, however, it has been thedifficulty in constructing cell lines that stably express Na_(v)subtypes that has slowed target-driven therapeutic design (Clare et al.(2000)).

SUMMARY OF INVENTION

[0007] Sodium channel alpha subunit expression was regulated in IMR-32cells by retinoic acid (RA). Quantitative PCR examination of IMR-32cells exposed to RA revealed up-regulation of endogenously expressedNa_(v)1.3 mRNA and down-regulation of other TTX-sensitive (TTX-S)Na_(v), including Na_(v)1.7 (FIG. 1). Western analysis indicated thatNav1.3 protein was expressed in IMR-32 cells maintained in RA (FIG. 3A).Thus, IMR-32 cells maintained in RA were ideally suited to screen formodulators of Na_(v)1.3 activity in cell-based assays includingveratridine-induced depolarization as measured by a voltage sensitivedye (FIG. 4). Quantitative PCR examination of IMR-32 cells culturedwithout RA revealed significant up-regulation of Na_(v)1.7 (SCN9a) mRNA(FIG. 1A). Western analysis indicated that Nav1.7 protein was expressedin IMR-32 cells cultured without supplements (FIG. 3B). Accordingly,these cells are ideally suited to screen for modulators of Na_(v)1.7activity in cell-based assays. The electrophysiological characteristicsof fast transient TTX-sensitive sodium currents expressed in cells withRA were consistent with the expression of SCN3a Na_(v) (FIGS. 5 to 7 and9). Inward currents in cells grown without RA were consistent with theexpression of Na_(v)1.7Na_(v) channels (FIG. 8). Na_(v)1.3 andNa_(v)1.7-specific antagonists are useful as therapeutic agents intreating human pathologies mediated (at least in part) by aberrantNa_(v)1.3 and Na_(v)1.7 expression. Manipulation of endogenous Na_(v)expression in IMR-32 cells by RA treatment in order to identify Na_(v)antagonists is a novel methodology that incorporates the followingprocedures:

[0008] 1) Undifferentiated IMR-32 cells were induced to differentiate byadding 9-cis RA (1 μM final) to normal IMR-32 growth medium, resultingin up-regulation of endogenous Na_(v) 1.3 expression and down-regulationof other TTX-S Na_(v), while culturing IMR-32 cells without RA wasemployed to maintain predominant expression of Na_(v) 1.7.

[0009] 2) Temporal changes in endogenous TTX-S Na_(v) expression wereassessed by quantitative PCR of cDNA templates synthesized from totalRNA of undifferentiated and RA-induced differentiated IMR-32 cells.

[0010] 3) RA-induced differentiated IMR-32 cells that predominantlyexpressed Na_(v) 1.3 were used to identify Na_(v) 1.3 channelantagonists in a cell-based assay.

[0011] 4) IMR-32 cells cultured without RA that predominantly expressedNa_(v)1.7 were used to identify Na_(v)1.7 channel antagonists in acell-based assay.

DESCRIPTION OF FIGURES

[0012]FIG. 1, Panels A and B:

[0013] Quantitative PCR data showing the effects of RA on the relativeexpression of Na_(v) mRNA.

[0014] Panel A: Predominant expression of Na_(v) 1.7 mRNA was observedin IMR-32 cells cultured without 9-cis-RA.

[0015] Panel B: 9-cis-RA (1 μM) induced up-regulation of Na_(v) 1.3 mRNAand down-regulation of Na_(v) 1.7 mRNA expression.

[0016]FIG. 2:

[0017] PCR determination of SCN beta subunit expression in IMR-32 cellscultured with (rRA+) and without (RA-) 9-cis RA (1 μm). Mixed cDNA(human brain, skeletal muscle and heart; invitrogen) served as apositive control for RT-PCR experiments. Intron-spanningoligonucleotides were used to distinguish cDNA-derived amplicons.

[0018]FIG. 3, Panels A and B:

[0019] Western immunoblot detection of Na_(v)1.3 and Na_(v)1.7expression in IMR 32 cells maintained under different growth conditions.

[0020] Panel A: IMR-32 cells maintained in media supplemented with 1 μMretinoic acid.

[0021] Specific detection of human Na_(v)1.3 protein isoforms by rabbitanti-rat Na_(v)1.3 antibody (Na_(v)1.3 Ig). Lane 1: Na_(v)1.3 Ig (1:300)recognized human Na_(v)1.3 protein isoforms of 215 kd, 160 kd, 135 kdand 115 kd. Lane 2: Pre-incubation of Na_(v)1.3 Ig (1:300) with ratNa_(v)1.3 peptide (1:1; mg:mg) blocked Na_(v)1.3 Ig recognition of humanNa_(v)1.3 protein isoforms of 215 kd, 160 kd, 135 kd and 115 kd. Lane 3:Pre-incubation of Na_(v)1.3 Ig (1:300) with rat Na_(v) Pan peptide (1:1;mg:mg) did not block Na_(v)1.3 Ig recognition of human Na_(v)1.3 proteinisoforms of 215 kd, 160 kd, 135 kd and 115 kd.

[0022] Panel B: IMR-32 cells maintained in media supplemented withoutretinoic acid. Specific detection of human Na_(v)1.7 protein isoforms byrabbit anti-rat PN1 antiboby (Na_(v)1.7 Ig). Lane 1: Na_(v)1.7 Ig(1:300) recognized human Na_(v)1.7 protein isoforms of 225 kd and 97 kd.Lane 2: Pre-incubation of Na_(v)1.7 Ig (1:300) with rat Na_(v)1.7peptide (1:1; mg:mg) blocked Na_(v)1.7 Ig recognition of human Na_(v)1.7protein isoforms of 225 kd and 97 kd. Lane 3: Pre-incubation ofNa_(v)1.7 Ig (1:300) with rat Na_(v) Pan peptide (1:1; mg:mg) did notblock Na_(v)1.7 Ig recognition of human Na_(v)1.7 protein isoforms of225 kd and 97 kd.

[0023]FIG. 4, Panels A, B and C:

[0024] Veratridine-induced depolarization of IMR-32 cells maintained inthe presence or absence of retinoic acid is blocked by tetrodotoxin(TTX) in a dose dependent manner using a voltage-sensitive dye-basedfluorometric assay (FLIPR™).

[0025] Panel A: The depolarization induced by veratridine stimulated Nainflux through Na channels in IMR-32 cells maintained in retinoic acidis shown as an increase in Fluorescence (F-F₀). TTX (100 nM; dottedline) or vehicle (solid line) was added on line at the first arrow andsix minutes later veratridine (10 μM final concentration) was added tostimulate Na influx through Na_(v) channels.

[0026] Panel B: TTX blocked veratridine-stimulated sodium influx in adose dependent manner in IMR-32 cells grown in the presence of RA. Datafrom a representative experiment is shown (IC₅₀=3.3 nM).

[0027] Panel C: TTX blocked veratridine-stimulated sodium influx in adose dependent manner in IMR-32 cells grown without RA. Data from arepresentative experiment is shown (IC₅₀=3.0 NM).

[0028]FIG. 5, Panels A and B:

[0029] Tetrodotoxin (TTX; 100 nm) blocked fast transient inward currentsin a population of IMR-32 cells grown in the presence of RA (1 μm).

[0030] Panel A: Voltage steps were applied between −80 and +50 mV inincrements of 10 mV from a holding potential of −100 mV. In this cell,inward currents were nearly completely blocked by TTX (92%). The TTXblockade was reversible upon washout of toxin.

[0031] Panel B: Current-voltage curves for the families of currentsshown in Panel A.

[0032]FIG. 6, Panels A and B:

[0033] The voltage dependence of steady state inactivation shifts to theleft in the presence of TTX (100 nM) in a population of IMR-32 cellsgrown in RA (1 μM).

[0034] Panel A: The membrane potential was held at −100 mV andsubsequently stepped for 500 msec to pre-pulse potentials rangingbetween −140 and −20 mV in increments of 10 mV prior to a voltage stepto 0 mV to elicit peak inward currents. TTX was not present.

[0035] Panel B: Plotted is the normalized peak current (I/Imax) obtainedat 0 mV after the indicated pre-pulse potential. The maximum peak inwardcurrent was elicited after pre-pulse potentials more negative than −110mV. Steady state inactivation curves are shown for a single cell in theabsence (solid squares) and presence (clear squares) of TTX (100 nM).The TTX-S current had a V_(0.5) more depolarized than the TTX-resistantcomponents of the inward current. The TTX-R inward currents in thesecells (RA+ as well as RA−) are largely composed of Cd²⁺ andmibefradil-sensitive Ca²⁺ currents.

[0036]FIG. 7, Panels A, B, C and D:

[0037] A second population of IMR-32 cells grown in RA was observed tohave little or no TTX sensitivity (100 nM).

[0038] Panel A: Family of currents was elicited by voltage stepsaccording to the protocol described in the previous figure.

[0039] Panel B: TTX (100 nM) had little effect on the inward currentselicited in this cell.

[0040] Panel C: Steady state inactivation in the absence of TTX wasdetermined according to the protocol described in the legend for FIG. 6.

[0041] Panel D: The voltage dependence of steady state inactivation wasnot significantly altered in the presence of TTX (100 nM) and theV_(0.5) was more negative than the TTX-S inward currents (−85 mV in thisexample).

[0042]FIG. 8, Panels A, B, C and D:

[0043] IMR-32 cells grown in the absence of RA were only partiallyblocked by TTX (100 nM). Cells were tested under conditions in which Cacurrents would be observed if present.

[0044] Panel A: Families of voltage activated currents was elicited byvoltage steps according to the protocol described in the previousfigures from a cell in the absence (left) and presence (right) of TTX.

[0045] Panel B: TTX (100 nM) slightly blocked the inward currentselicited in this cell and shifted the voltage dependence of activationto the left.

[0046] Panel C: Steady state inactivation in the absence of TTX wasdetermined according to the protocol described in FIG. 5.

[0047] Panel D: The voltage dependence of steady state inactivationunder control conditions revealed an inactivating current withV_(0.5)=−81 mV.

[0048]FIG. 9, Panels A, B, and C:

[0049] Panel A: The proportion of TTX-sensitive inward currentsexpressed in IMR-32 cells grown with RA (solid black squares) wasdirectly related to the proportion of inward currents having adepolarized V_(0.5) for steady state inactivation. IMR-32 cells grown inthe absence of RA expressed TTX-R currents with V_(0.5)˜−85 mV. IMR-32RA+ cells expressing large TTX-S currents were those that revealed avoltage dependence of steady state inactivation similar to that reportedfor SCN3a recombinant channels (˜−65 mV (Cummins et al. (2001)), 70 mV(Chen et al. (2000)).

[0050] Panel B: The electrophysiological characteristics of the currentin cells that do not have a preponderance of Nav1.3 ion channels isshown.

[0051] Panel C: The electrophysiological characteristics of the currentin cells that is consistent with expression of Nav1.3 ion channels isshown.

DETAILED DESCRIPTION OF THE INVENTION

[0052] The present invention provides methods to identify compounds thatmodulate the function of endogenously expressed ion channels, including,but not limited to, Na_(v) 1.3 and Na_(v) 1.7. The present inventionprovides IMR-32 cells differentiated by 9-cis RA predominantlyexpressing Na_(v) 1.3 and thus, are ideally suited to identify compoundsthat modulate Na_(v) 1.3 function.

[0053] Accordingly, we approached this challenge by first determiningwhether Na_(v)1.3 and Na_(v)1.7 function could be effectively studied ina stable cell line. Specifically, we examined Na_(v) 1.3 and Na_(v)1.7function in the human neuroblastoma cell line, IMR-32, under differentgrowth conditions. The IMR-32 neuroblastoma cell line (ATCC #CCL-127)was established by W. W. Nichols, J. Lee and S. Dwight in April, 1967from an abdominal mass occurring in a 13-month-old Caucasian male(Tumilowicz et al., “Definition of a Continuous Human Cell Line Derivedfrom Neuroblastoma”, Cancer Res. (1970) 30:2110-2118). The tumor wasdiagnosed as a neuroblastoma with rare areas of organoid differentiationand the cell line contains two cell types: a small neuroblast-like cell(predominant) and a large hyaline fibroblast. Initially, DNA microarrayanalysis was used to identify IMR-32 as a cell line that potentiallyexpressed Na_(v) 1.3 and Na_(v) 1.7. Through the use of quantitativePCR, electrophysiological recordings and Na_(v)1.3 and Na_(v)1.7specific antibodies, we demonstrated that IMR-32 cells do, in fact,express endogenous TTX-sensitive inward currents and predominantlyNa_(v) 1.3 mRNA when cultured in the presence of RA (1 μM) orpredominantly Na_(v) 1.7 mRNA when cultured without RA (1 μM). Nav1.3and Nav1.7 were detected in Western blot analysis indicating thetranslation into protein in this line under different cultureconditions. Thus, both RA-differentiated and undifferentiated IMR-32cells are ideal for use in vitro assays designed to investigate humanNa_(v) 1.3 and Na_(v) 1.7 function. As such, this allows a target drivenapproach to be taken with regard to Na_(v) 1.3 and Na_(v) 1.7 drugdiscovery.

[0054] Candidate compounds identified using the methods of the presentinvention are also useful for treating diseases and conditions mediatedby Na_(v) 1.3 and Na_(v)1.7 including, but not limited to, neuropathicpain, chronic pain, anxiety, seizure, epilepsy (up-regulation inepileptic hippocampus tissue (Whitaker et al. (2001)), ischemia,migraine, bipolar disorder, deafness, schizo-affective disorder,Alzheimer's disease, stroke, Parkinson's disease, tinnitus, depressionand substance abuse, asthma and chronic stress, prostate cancer andother cancerous tissues expressing high levels of Nav1.3 and Nav1.7.Compounds are administered to a subject in need thereof as an activeingredient in a suitable pharmaceutical composition.

[0055] The compounds of the present invention may be any type of organicor inorganic substances, including, but not limited to, proteins,peptides, antibodies, small organic molecules and inorganic molecules.

[0056] Pharmaceutically useful compositions comprising modulators ofNa_(v) 1.3 and/or Na_(v) 1.7 activity, may be formulated according toknown methods such as by the admixture of a pharmaceutically acceptablecarrier. Examples of such carriers and methods of formulation may befound in Remington's Pharmaceutical Sciences. To form a pharmaceuticallyacceptable composition suitable for effective administration, suchcompositions will contain an effective amount of the protein, DNA, RNA,or modulator compound.

[0057] The pharmaceutical compositions of the present invention can beprepared according to conventional pharmaceutical techniques. Apharmaceutically acceptable carrier may be used in the compositions ofthe present invention. A wide variety of pharmaceutical compositions aresuitable for use in the present invention. It is readily apparent tothose skilled in the art that different compositions may be useddepending on the route of administration including, but not limited to,intravenous (both bolus and infusion), oral, nasal, pulmonary,transdermal, topical with or without occlusion, intraperitoneal,intracranially, epidurally, directly into CSF, subcutaneous,intramuscular, intrathecal, ocular, or parenteral, all well known tothose of ordinary skill in the pharmaceutical arts. In preparing thecompositions in oral dosage form, one or more of the usualpharmaceutical carriers may be employed, such as water, glycols, oils,alcohols, flavoring agents, preservatives, coloring agents, syrup andthe like in the case of oral liquid preparations (for example,suspensions, elixirs and solutions), or carriers such as starches,sugars, diluents, granulating agents, lubricants, binders,disintegrating agents and the like in the case of oral solidpreparations (for example, powders, capsules and tablets).

[0058] Alternatively, the compounds may be administered parenterally viainjection of a formulation consisting of the active ingredient dissolvedin an inert liquid carrier. Acceptable liquid carriers include vegetableoils such as peanut oil, cotton seed oil, sesame oil, and the like, aswell as organic solvents such as solketal, glycerol formal, and thelike. As an alternative, aqueous parenteral formulations may also beused. For example, acceptable aqueous solvents include water, Ringer'ssolution and an isotonic aqueous saline solution. Further, a sterile,non-volatile oil can usually be employed as solvent or suspending agentin the aqueous formulation. The formulations are prepared by dissolvingor suspending the active ingredient in the liquid carrier such that thefinal formulation contains from 0.005 to 10% by weight of the activeingredient. Other additives including a preservative, an isotonizer, asolubilizer, a stabilizer and a pain-soothing agent may adequately beemployed.

[0059] The compounds may be administered ocularly via application of aformulation consisting of the active ingredient dissolved in an inertaqueous liquid carrier. Such aqueous liquid formulations are useful, forexample, in the treatment of diabetic retinopathy. Acceptable aqueoussolvents include water, Ringer's solution, and an isotonic aqueoussaline solution. The formulations are prepared by dissolving orsuspending the active ingredient in the liquid carrier such that thefinal formulation contains from 0.005 to 10% by weight of the activeingredient. Other additives including a preservative, an isotonizer, asolubilizer, a stabilizer and a pain-soothing agent may adequately beemployed.

[0060] The compounds of the present invention may also be administeredin the form of liposome delivery systems, such as small unilamellarvesicles, large unilamellar vesicles and multilamellar vesicles.Liposome delivery systems, are well known in the art, and may be formedfrom a variety of phospholipids, such as cholesterol, stearylamine orphosphatidylcholines.

[0061] As used herein, a “therapeutically effective amount” of theinstant pharmaceutical composition, or compound therein, means an amountthat is effective in treating a disease or condition medicated at leastin part by Na_(v) 1.3 and/or Na_(v) 1.7, neuropathic pain, chronic pain,anxiety, seizure, epilepsy, ischemia, migraine, bipolar disorder,deafness, schizo-affective disorder, Alzheimer's disease, stroke,Parkinson's disease, tinnitus, depression and substance abuse, prostatecancer, asthma, and chronic stress. The instant pharmaceuticalcomposition will generally contain a per dosage unit (e.g., tablet,capsule, powder, injection, teaspoonful and the like) from about 0.001to about 100 mg/kg. In one embodiment, the instant pharmaceuticalcomposition contains a per dosage unit of from about 0.01 to about 50mg/kg of compound, but preferably from about 0.05 to about 20 mg/kg.Methods are known in the art for determining therapeutically effectivedoses for the instant pharmaceutical composition. The effective dose foradministering the pharmaceutical composition to a human, for example,can be determined mathematically from the results of animal studies.Furthermore, compounds of the present invention can be administered inintranasal form via topical use of suitable intranasal vehicles, or viatransdermal routes, using those forms of transdermal skin patches wellknown to those of ordinary skill in the art. To be administered in theform of a transdermal delivery system, the dosage administration will,of course, be continuous rather than intermittent throughout the dosageregimen.

[0062] Oral Dosage Forms

[0063] Because of their ease of administration, tablets and capsulesrepresent an advantageous oral dosage unit form, wherein solidpharmaceutical carriers are employed. If desired, tablets may besugar-coated or enteric-coated by standard techniques. For liquid forms,the active drug component can be combined in suitably flavoredsuspending or dispersing agents such as the synthetic and natural gums,for example, tragacanth, acacia, methyl-cellulose and the like. Otherdispersing agents that may be employed include glycerin and the like.

[0064] The present invention is also directed to methods for screeningtest compounds that are suspected of being able to modulate theexpression of DNA or RNA encoding Na_(v) 1.3 and/or Na_(v) 1.7 as wellas the function of the Na_(v) 1.3 and/or Na_(v) 1.7 ion channels invitro or in vivo. Compounds that modulate these activities may includebut are not limited to DNA, RNA, peptides, proteins, ornon-proteinaceous organic or inorganic molecules. Compounds may modulateby increasing or attenuating the expression of DNA or RNA encodingNa_(v) 1.3 and/or Na_(v) 1.7, or the function of the Na_(v) 1.3 and/orNa_(v) 1.7 ion channels. Compounds that modulate the expression of DNAor RNA encoding Na_(v) 1.3 and/or Na_(v) 1.7 or the function of Na_(v)1.3 and/or Na_(v) 1.7 protein may be detected by a variety of assaysutilizing cells or fractions and components thereof, prepared accordingto the methods disclosed in present specification or standard methodswell known to those skilled in the art. The assay may be a simple“yes/no” assay to determine whether there is a change in expression,ligand binding, or function of the target molecule. The assay may bemade quantitative by comparing the expression, ligand binding orfunction of the target molecule in the presence of a test sample withthe levels of expression, ligand binding, or function of the targetmolecule in a standard or control sample. Modulators identified in thisprocess are useful as therapeutic agents, research tools, and diagnosticagents. Such modulators can include agonists, antagonists, and inverseagonists of the Na_(v) 1.3 and/or Na_(v) 1.7 ion channels.

[0065] It is to be understood that this invention is not limited to theparticular methodologies, protocols, constructs, formulae and reagentsdescribed and as such may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention.

[0066] It must be noted that as used herein and in the appended claims,the singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference to“a gene” is a reference to one or more genes and includes equivalentsthereof known to those skilled in the art, and so forth.

[0067] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this invention belongs. Although any methods,devices, and materials similar or equivalent to those described hereincan be used in the practice or testing of the invention, the preferredmethods, devices and materials are now described.

[0068] All publications and patents mentioned herein are incorporatedherein by reference for the purpose of describing and disclosing, forexample, the constructs and methodologies that are described in thepublications, which might be used in connection with the presentlydescribed invention. Nothing herein is to be construed as an admissionthat the inventor is not entitled to antedate such disclosure by virtueof prior invention.

[0069] The following Examples are provided for the purpose ofillustrating the present invention, without, however, limiting thepresent invention to the specific disclosure contained in the followingExamples.

EXAMPLE 1 Exposure Of IMR-32 Cells to RA Alters the Expression of aPopulations of TTX-S Sodium Channel Alpha Subunits Revealing anUp-Regulation of Na_(v)1.3 and Down-Regulation Of Na_(v)1.7 mRNA

[0070] Cell Culture

[0071] IMR-32 cells (American Tissue Culture Collection #CCL-127,Manassas, Va.), stored at −140° C. in freezing media (90% fetal bovineserum, 10% DMSO), were rapidly-thawed at 37° C., washed in normal IMR-32medium (Eagle's minimum essential media containing Hanks balanced salts,1.5 gram/liter sodium bicarbonate, 1 mM sodium pyruvate, 0.1 mMnon-essential amino acids, 2 mM L-glutamine, 10% fetal bovine serum) toremove the DMSO and centrifuged at 1000 rpm for two minutes at 4° C. topellet the cells. The cell pellet was re-suspended in fresh IMR-32medium, plated in a 150 cm² tissue culture flask and incubated at 37° C.in 5% CO₂ until confluent. Confluent cells were washed in calcium-freephosphate buffer, treated with 0.02% trypsin until dislodged andserially-passed into 150 cm² tissue culture flasks containing IMR-32media supplemented with 9-cis RA to 1 μM final concentration andincubated as above. Initially, IMR-32 cells cultured in 9-cis RAproliferated slowly. Following a three-week culture in RA, the cellsstabilized and were passed 1:2 to maintain robust proliferation. Cellsdestined for use in the fluorescence assay were passed usingcalcium-free buffer (Versene; Gibco) to ensure retention of TTXsensitivity.

[0072] (note: copy # PCR wasn't performed for the betas) PCR-basedscreening of IMR-32 cells was done to determine mRNA levels of alpha andbeta sodium channel subunits. Quantitative PCR was used to determinerelative expression of TTX-sensitive voltage-gated sodium channels inIMR-32 cells since veratridine-induced depolarization was completelysuppressed by 100 nM TTX (FIG. 3). For this procedure, IMR-32 cells weregrown in 10 cm² culture dishes until 80% confluent; whereupon, total RNAwas isolated from the cells with Trizol reagent (Gibco) as per themanufacturer's protocol. Following spectrophotometric quantification,1.5 μg of total RNA, isolated from IMR-32 cells grown either in thepresence or absence of 9-cis RA (1 μM) were reverse-transcribed intocDNA with Superscript II reverse transcriptase (Gibco) as per themanufacturer's protocol. Synthesized cDNAs were diluted 1:4 innuclease-free H₂O supplemented with poly-inosine to a finalconcentration of 50 nanograms per milliliter, heated at 70° C. for fiveminutes and placed on ice for an additional two minutes. Primers thatspan putative introns were used in PCR experiments to permit positiveidentification of amplicons synthesized from cDNA templates. Primersequences included: Na_(ν) 1.1 (Forward) 5′ CAA AAG CCT ATA CCT CGA CCA3′ SEQ ID NO:1 (Reverse) 5′ TCA GCT CGG CAA GAA ACA TAC 3′ SEQ ID NO:2Na_(ν) 1.2 (Forward) 5′ ACT GGT TAG CTT AAC TGC AAA TGC CTT GG 3′ SEQ IDNO:3 (Reverse) 5′ ACG CTT ACA TCA AAC ATC TCT CCA GTG G 3′ SEQ ID NO:4Na_(ν) 1.3 (Forward) 5′ TTG GAA GAA GCA GAA CAA AAA GAG G 3′ SEQ ID NO:5(Reverse) 5′ AGG GGA GCA GAA TTT TTT GTC ACT GG 3′ SEQ ID NO:6Na_(ν) 1.4 (Forward) 5′ TCT CAG AGC CTG AGG ATA GCA 3′ SEQ ID NO:7(Reverse) 5′ AAT GAC TCG CCG CTG CTC AAT 3′ SEQ ID NO:8 Na_(ν) 1.6(Forward) 5′ TTG GAG TAT TTC TCC CTC TGA G 3′ SEQ ID NO:9 (Reverse)5′ ATG CAG CTT CAG TAT ACA TTC CA 3′ SEQ ID NO:10 Na_(ν) 1.8 (Forward)5′ TGT GGA ACA GCC TGA GGA ATA CTT GG 3′ SEQ ID NO:11 (Reverse) 5′ TGGAGG GGA TGG CGC CCA CCA AGG 3′ SEQ ID NO:12 Na_(ν) 1.9 (Forward) 5′ ATCCCT TCG GAC ACT GAG AGC TTT AAG ACC 3′ SEQ ID NO:13 (Reverse) 5′ TGG GCTGCT TGT CTA CAT TAA CAG AAT CC 3′ SEQ ID NO:14

[0073] Amplicons were fractionated by ethidium bromide agarose gelelectrophoresis and visualized under ultraviolet light. Amplicons of thepredicted molecular weight were subcloned into the pCR4-TOPO TA cloningvector (Invitrogen, Carlsbad, Calif.) as per the manufacturer's protocoland sequenced. Relative TTX-sensitive Na_(v) expression in IMR-32 cellswas determined by quantitative PCR using sequence-positive plasmids asstandards. As shown in FIG. 1, expression of Na_(v) 1.7 (SCN9a) mRNApredominates in undifferentiated IMR-32 cells. IMR-32 cells cultured formore than three weeks in 1 μM 9-cis RA predominantly express Na_(v) 1.3mRNA with concomitant down-regulation of Na_(v) 1.7 (Panel B).Na_(v)1.1, Na_(v)1.2, Na_(v)1.4 and Na_(v)1.6 mRNA were not detected byRT-PCR.

[0074] Expression of Na channel beta subunits 1 to 3 in IMR-32 cellsgrown with or without RA was determined using non-quantitative RT-PCR.Primer sequences were: SCN1b SEQ ID NO:15 (Forward)5′ ACGCTGAGACCTTCACCGAGT 3′ SEQ ID NO:16 (Reverse)5′ ACCACAACACCACAATGAGCAC 3′ SCN2b SEQ ID NO:17 (Forward)5′ GACTAACATCTCAGTCTCTGAAAAT 3′ SEQ ID NO:18 (Reverse)5′ GGCTGCACGTTTCTCAGCATCA 3′ SCN3b SEQ ID NO:19 (Forward)5′ TGACTACCTTGCCATCCCATCT 3′ SEQ ID NO:20 (Reverse)5′ CTTCTCAGTTCTGGCAGAGTCTTA 3′

[0075] Mixed cDNA (human brain, skeletal muscle and heart; Invitrogen)served as a positive control for PCR experiments. Intron-spanningoligonucleotides were used to distinguish cDNA-derived amplicons. Beta3,but not beta1 and beta2, was detected in IMR-32 cells exposed to RA.Beta2 and beta3 were detected in cells not exposed to RA.

EXAMPLE 2 Cell-Based Assay Using Voltage Sensitive Dyes to MeasureNa_(v) Activity in IMR-32 Cells

[0076] IMR-32 cells cultured in growth medium (Eagles minimal essentialmedia, 2 mM L-glutamine and Earle's BSS adjusted to contain 1.5 g/Lsodium bicarbonate, 0.1 mM non-essential amino acids, 1.0 mM sodiumpyruvate, 1 μM RA) are plated (40 μl per well) at a density of 5.5×10⁶cells per 384-well plate and incubated for eighteen to twenty-four hoursat approximately 37° C. in 5% CO₀. IMR-32 cells were plated on tissueculture-treated plates without poly-D-Lysine since prolonged exposure topoly-D-lysine (e.g. commercially prepared cell culture plates) reducedcell adhesion and viability. The saline used in most studies was 2K/2Cabuffer, and it contained (in mM): 130 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 2mM CaCl₂, 20 mM HEPES, pH 7.4. On the day of the assay, 20×voltage-sensitive dye (Molecular Devices, Sunnyvale, Calif.; catalog #R8034) was diluted 1:10 in 2K/2Ca saline supplemented with bariumchloride (250 μM final concentration for Na_(v)1.3 expressing cells; 375μM final concentration for predominantly Na_(v)1.7-expressing cells) andadded (40 μl per well) to the cells without mixing. The cells wereincubated in voltage-sensitive dye for five to sixty minutes at roomtemperature in the dark; whereupon, the cells were challenged on-linewith test compounds suspected of having Na_(v) 1.3 and/or Na_(v) 1.7modulating activity, using a fluorometric imaging plate reader (FLIPR™)for compound addition and data collection. In this assay, 13 μl of eachtest compound (170 μM initial) were added with mixing (10 μl/sec) toeach well and incubated for six minutes. Subsequently, 5 μl of 200 μMveratridine (Na_(v) ‘activator’) were added with mixing (15 μl/sec) toeach well to achieve a final veratridine concentration of 10 μM. Cellfluorescence was monitored for an additional 70 seconds. Depolarizationelicited by the influx of Na⁺ ions produced an increase in fluorescence.The observed depolarization induced by veratridine was dependent onexternal Na⁺. The screening window index W [where W=3 * (sd unblockedsignal+sd blocked signal)/(unblocked mean signal−blocked mean signal)]was determined by including 100 nM TTX in half the wells of a 384-platewith subsequent stimulation of all wells with veratridine. For thisexample, the W value ranged from 0.66 to 0.77.

[0077]FIG. 4, Panel A shows an example of veratridine-induced Na_(v)activation following six minutes pre-incubation with either 2K/2Casaline (solid line) or 100 nM TTX (dashed line) in IMR-32 cells that hadbeen maintained in RA. A dose-dependent block by the neurotoxin TTX wasobserved. The concentration to half block the veratridine-inducedincrease in fluorescence (IC₅₀)=2.8+/−0.3 nM (n=3 separate experiments,mean +/−SD) (FIG. 3, Panel B). Final DMSO concentrations up to 2% weretolerated. TTX blockade was also examined in IMR-32 cells grown withoutRA and the IC₅₀ was 2.6+/−0.6 nM (n=2) (FIG. 3, Panel C). The steepnessof the dose response relationship appeared to be less in cultures grownwithout RA (1.0, 1.5) vs. with RA (0.76, 0.89 and 0.79). Furthermore,TTX had a tendency to completely block a greater proportion of theveratridine-induced response in cells that were maintained with RA (88,89, 90%) compared to those grown in the absence of RA (82, 80%).

[0078] In another example, cells were washed with 0-Na solutions (150 mMTMA-Cl, 0.1 mM CaCl₂, 1.2 mM MgCl₂, 10 mM Dextrose, 10 mM HEPES freeacid, pH to 7.4 using CsOH), incubated in CC2 (Aurora Bioscience) for 30min and subsequently in DiSBAC2 (Aurora Bioscience) together withveratridine, and assayed using ViPR™ technology. Addition ofNa+-containing solution (similar volume) caused a depolarization of themembrane potential and decreased FRET between the CC2 and oxonol dyes.Antagonists were incubated together with veratridine/DiSBAC2. TTXproduced dose dependent decreases in the FRET signal (data not shown).

EXAMPLE 3 Exposure of IMR-32 Cells to RA Alters the Expression ofPopulations of TTX-S Sodium Currents Consistent with an Up-Regulation ofNa_(v) 1.3.

[0079] Electrophysiological Recordings from IMR-32 Cells

[0080] The endogenous voltage gated Na currents expressed in IMR-32cells grown in the presence or absence of 1 μM RA were measured usingstandard whole cell voltage clamp techniques (Hamill et al. (1981). Thewhole cell patch clamp technique was used to record voltage-activatedcurrents from IMR-32 cells maintained for two or more days on 12 mmglass coverslips in the presence or absence of RA (1 μM). Cells werevisualized using a Nikon Diaphot 300 with DIC Nomarski optics. Thestandard physiological saline (1Ca tyrodes (“Tyrodes”) contains: 130 mMNaCl, 4 mM KCl, 1 mM CaCl₂, 1.2 mM MgCl₂, and 10 mM hemi-Na-HEPES (pH7.3, 295-300 mOsm as measured using a Wescor 5500 vapor-pressure(Wescor, Inc., Logan, Utah)). Recording electrodes are fabricated fromborosilicate capillary tubing (R6; Garner Glass, Claremont, Calif.), thetips are coated with dental periphery wax (Miles Laboratories, SouthBend, Ind.), and have resistances of 1 to 2 MΩ when containing anintracellular saline designed to block outward currents: 140 mM CsCl,0.483 mM CaCl₂, 2 mM MgCl₂, 10 mM HEPES free acid and 1 mM K₄-BAPTA (100nM free Ca⁺²); pH 7.4, with dextrose added to achieve 290 mOsm). Currentand voltage signals are detected and filtered at 2 kHz with an Axopatch1D patch-clamp amplifier (Axon Instruments, Foster City, Calif.),digitally recorded with a DigiData 1200B laboratory interface (AxonInstruments) and PC compatible computer system and stored on magneticdisk for off-line analysis. Data acquisition and analysis are performedwith PClamp software.

[0081] The total membrane capacitance (C_(m)) was determined as thedifference between the maximum current after a 30 mV hyperpolarizingvoltage ramp from −100 mV (generated at a rate of 10 mV/ms) and thesteady state current at the final potential (−130 mV) (Dubin et al.(1999)).

[0082] Families of voltage-gated inward currents were obtained using astandard P/−4 protocol from −100 mV. Depolarizing voltage steps inincrements of 10 mV were applied from a holding potential of −100 mV.Steady state inactivation was elicited by measuring the peak current at0 mV after a 500 msec pre-pulse voltage between −140 and −20 mV inincrements of 10 mV.

[0083] Cells grown for more than two weeks in RA and tested in Tyrodesrevealed larger peak inward currents (−75.3+/−10.6 pA/pF (n=24) vs.−43.3+/−6.1 pA/pF (n=6)) compared to cells cultured without RA (p<0.02,Student's t test). The cell sizes were similar in the presence andabsence of RA (10.2 vs. 11.6 pF; p=0.558). Cells were thoroughly rinsedin Tyrodes without RA prior to recording. Inward currents were reducedin low Na Tyrodes where TMA was substituted for the majority of Na (25mM). Under the conditions used in these studies (with CsCl in the pipet)outward currents were blocked (FIGS. 5-8).

[0084] Inward currents elicited in IMR-32 cells grown in RA revealedheterogeneity in their sensitivity to bath applied TTX at 100 nM (FIGS.5 and 7). The concentration of TTX chosen for the electrophysiologicalstudies (100 nM) completely blocked the veratridine-induced fluorescencesignal in the assay described in Example 3. In some cells, inwardcurrents were substantially and reversibly blocked by TTX (FIG. 5).These cells had complex steady state inactivation curves and TTX blockedcurrents that inactivated only at depolarized potentials, having avoltage dependence V_(0.5) consistent with that reported for therecombinant human SCN3a (−58 and −69 mV (Cummins et al. (2001)), andnative rat sodium currents in axotomized small DRG neurons (−72 mV)(Cummins et al. (2001)). Inward currents in other cells from the sameplating of IMR-32 cells with RA were largely TTX-R (FIG. 7).Interestingly, steady state inactivation relationships revealed thatthis latter population of cells lacked or expressed less of the inwardcomponent contributing to the depolarized V_(0.5) (FIG. 7, Panels C andD) The voltage to half-inactivate the channels was near −85 mV. Themolecular identity of the TTX-R inward current included, in large part,calcium currents since the TTX-R current could be blocked nearlycompletely by 500 μM Cd²⁺ and in part by 5 μM mibefradil. Cd²⁺ blocked50+/−2% and 68+/−6% of the TTX-R current at 100 and 500 μM,respectively.

[0085] There was a strong correlation between the degree of TTX blockand the magnitude of the inward current component with a depolarizedsteady state voltage to half inactivation (V_(0.5)) (FIG. 9 Panel A).Thus, inward currents that were largely TTX-S revealed a voltagedependence for steady state inactivation that was shifted to the rightalong the voltage axis, consistent with the functional expression ofSCN3a channels.

[0086] The TTX-S component of inward currents expressed in IMR-32 cellsnot exposed to RA tended to be a smaller proportion of total currentcompared to that in RA-treated cells (17+/−6% (n=6) vs. 36+/−7(n=19)).Fast transient calcium currents contributed to the TTX-R component incells cultured without RA as well. IMR-32 cells grown without RAexpressed fast transient inward currents with a negative shifted voltagedependence of inactivation compared to sister cultures maintained in RA.The V_(0.5) for steady state inactivation is similar to the valuesdetermined for PN1 (SCN9a, Na_(v)1.7) expressed in a recombinantexpression system (Sangameswaran et al. (1997); Cummins et al. (1998)).

[0087] Thus, IMR-32 cells showed heterogeneity in their block by TTX inelectrophysiological (FIGS. 5 to 8) assays. Since TTX nearly completelyblocks the veratridine induced signal in both RA+ and RA− treated cells,the depolarization observed in the fluorescence assay requiresactivation of Nav but other secondary conductances may contribute ifactivated by the veratridine-induced depolarization.

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What is claimed is:
 1. A method for inducing expression of Na_(v) 1.3ion channels from IMR-32 cells comprising culturing the IMR-32 cellswith a sufficient amount of a differentiating agent for a sufficientperiod of time for the IMR-32 cells to produce the Na_(v) 1.3 ionchannels.
 2. The method of claim 1 wherein the differentiating agent isretinoic acid or a growth factor.
 3. A method for identifying testcompounds that modulate Na_(v) 1.3 ion channels comprising contactingthe cells of claim 1 with a test compound and measuring an effect ofsodium ion influx on the IMR-32 cells, wherein said effect results fromincreased or decreased influx of sodium ions in the presence or absenceof a known Na_(v) 1.3 ion channel agonist or antagonist.
 4. The methodof claim 2 wherein the growth factor is a nerve growth factor.
 5. Acompound identified in the method of claim 3 as being a modulator ofNa_(v) 1.3 ion channels wherein said compound was not previously knownto have Na_(v) 1.3 ion channel modulating activity.
 6. A method fortreating a disease or condition mediated by Na_(v) 1.3 ion channelscomprising administration of an effective amount of a pharmaceuticalcomposition comprising a Na_(v) 1.3 modulator compound according toclaim 5, to a patient in need of such treatment.
 7. The method of claim6 wherein the disease or condition is neuropathic pain, chronic pain,anxiety, seizure, epilepsy, ischemia, migraine, bipolar disorder,deafness, schizo-affective disorder, Alzheimer's disease, stroke,Parkinson's disease, tinnitus, depression and substance abuse, prostatecancer, asthma, or chronic stress.
 8. A cell line expressing Na_(v) 1.7ion channels wherein said cell line is IMR-32.
 9. A method foridentifying test compounds that modulate Na_(v) 1.7 ion channelscomprising contacting the cells of claim 8 with a test compound andmeasuring an effect of sodium ion influx on the IMR-32 cells, whereinsaid effect results from increased or decreased influx of sodium ions inthe presence or absence of a known Na_(v) 1.7 ion channel agonist orantagonist.
 10. A compound identified in the method of claim 9 as beinga modulator of Na_(v) 1.7 ion channels wherein said compound was notpreviously known to have Na_(v) 1.7 ion channel modulating activity. 11.A method for treating a disease or condition mediated by Na_(v) 1.7 ionchannels comprising administration of an effective amount of apharmaceutical composition comprising a Na_(v) 1.7 modulator compoundaccording to claim 10, to a patient in need of such treatment.
 12. Themethod of claim 11 wherein the disease or conditions is neuropathicpain, chronic pain, anxiety, seizure, epilepsy, ischemia, migraine,bipolar disorder, deafness, schizo-affective disorder, Alzheimer'sdisease, stroke, Parkinson's disease, tinnitus, depression and substanceabuse, prostate cancer, asthma, and chronic stress.